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Oligonukleotidi

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Oligonukleotidi ni molekuli fupi za Asidi DeoksiriboNukleini (ADN) au Asidi RiboNukleini (ARN), zilizo na matumizi mbalimbali kama vile katika upimaji wa vinasaba, utafiti na biolojia ya ujasusi. Molekuli hizi hutengenezwa katika maabara kwa usanisi wa kemikali wa solid-phase. [1] Aidha, zinaweza kutengenezwa kama molekuli zenye strandi moja kwa kuzingatia mfuatano ulioagizwa na mtumiaji,hivyo basi ni muhimu katika usanisi wa jeni bandia, polymerase mnyororo mmenyuko (PCR), utambuzi mpangilio wa DNA, molecular cloning na kama molecular probes . Katika viumbehai, oligonukleotidi hupatikana kama molekuli ndogo za RNA zinazotumika katika udhibiti wa utengenezaji wa protini kutokana na taarifa iliyopo kwenye jeni (km microRNA ).[2]

Oligonukleotidi huundwa kwa mpangilio maalum wa nukleotidi . Urefu wa molekuli hizi huonyeshwa kwa " -mer " (kutoka kwa Kigiriki, meros, "sehemu"). Kwa mfano, oligonukleotidi ya nukleotidi (nt) sita ni heksama, ilhali ya ishirini na tano kwa kawaida huitwa "25-mer". Oligonukleotidi huambata oligonukleotidi zingine mfano wa DNA au RNA , kwa utaratibu mahususi, ili kuunda dupleksi. Sifa hii ya oligonukleotidi ndio msingi wa matumizi yake kama probes katika kugundua mfuatano maalum wa DNA au RNA. Mifano ya taratibu zinazotumia oligonukleotidi ni pamoja na DNA microarray, Southern blot, uchambuzi wa oligonukleotidi antisensi, [3] fluorescent in situ hybridization ( FISH), PCR, na usanisi wa jeni bandia.

Oligonukleotidi huundwa kwa 2'-deoksiribonukleotidi (oligodeoksiribonukleotidi), ambazo zinaweza kurekebishwa kwenye uti wa mgongo au kwenye nafasi ya 2' ya sukari ili kuafikia athari tofauti tofauti za kifamasia. Marekebisho haya hupatia oligonukleotidi sifa mpya na kuzifanya kuwa kipengele muhimu katika tiba ya antisense . [4] [5]

Usanisi

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Oligonukleotidi hutengenezwa kwa usanisi kemikali unaotumia vitalu vya ujenzi kama vile protected phosphoramidites za asili au nukleosidi zilizoboreshwa kikemikali. Ujenzi wa mnyororo wa oligonukleotidi huendelea katika mwelekeo wa 3' hadi 5' kwa kufuata utaratibu wa kawaida unaojulikana kama "synthesis cycle". Kukamilika kwa mzunguko mmoja wa sintetiki husababisha kuongezwa kwa nukleotidi kwenye mnyororo unaokua. Mavuno chini ya 100% ya kila hatua ya syntetiki na tukio la mmenyuko ya upande huwa athari katika ufanisi wa mchakato huu. Kwa kawaida, mlolongo wa oligonukleotidi huwa mfupi (urefu wa nukleotidi 13-25 ). [6] Nukleotidi 200, mara nyingi, huwa urefu wa juu zaidi wa oligonukleotidi syntetiki. HPLC na njia zingine zinaweza kutumika kutenga oligonukleotidi zilizo na mlolongo wa nukleotidi unaofaa.

Marekebisho ya kemikali

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Kuunda oligonukleotidi fupi fupi zenye uimara wa kemikali ndiyo iliyokuwa changamoto ya kwanza katika kutengeneza tiba za ASO. Oligonukleotidi za asili huharibiwa kwa urahisi na nucleases, vimeng'enya ambavyo hupasua nukleotidi na vinapatikana kwa wingi katika kila aina ya seli. [7] Oligonukleotidi fupi pia huwa na mshikamano dhaifu, jambo ambalo huchangia kwa kuharibiwa kwake in vivo. [8]

Marekebisho ya uti wa mgongo

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Nukleotidi za nukleosidi organothiophosphate (PS) analogs hupatia oligonukleotidi baadhi ya sifa za manufaa. Sifa hizi kuu zinazotokana na uti wa mgongo wa PS kwenye nukleotidi ni utambuzi wa diastereomer kwa kila nukleotidi na uwezo wa kufuatilia kwa urahisi mimenyuko inayohusisha nukleotidi za phosphorothioate, ambayo ni muhimu katika usanisi wa oligonukleotidi. [9] Maboresho ya uti wa mgongo wa PS hulinda oligonukleotidi dhidi ya uharibifu usiohitajika na vimeng'enya. [10] Uboreshaji wa uti wa mgongo wa nukleotidi hutumiwa sana kwa ajili ya kuafikiwa kwake kwa urahisi na usahihi kwenye nukleotidi nyingi. [9] Kuunganishwa kwa fluorescent kwenye mwisho wa 5' na 3' ya oligonukleotidi yaliripotiwa kutathmini miundo, mienendo na mwingiliano wake kwenye mazingira. [11]

Marekebisho ya pete ya sukari

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Uboreshaji mwingine ambao ni muhimu katika matumizi ya matibabu ya oligonukleotidi ni marekebisho ya 2' ya sukari . Kurekebisha sukari katika nafasi ya 2' huongeza ufanisi wa oligonukleotidi kwa kuongeza uwezo wa kufungamana na oligonukleotidi lengwa, haswa katika tiba za oligonukleotidi antisensi . [8] Pia hupunguza mshikamano na protini zisizo maalum, na kuongeza usahihi wa uhusiano na protini maalum lengwa . [8] Marekebisho mawili kati ya yanayotumika sana ni 2'-O-methyl na 2'-O-methoxyethyl. [8] Marekebisho ya fluorescent kwenye nukleobesi pia yaliripotiwa. [11]

Oligonukleotidi Antisensi

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Oligonukleotidi Antisensi (ASO) ni strandi moja za DNA au RNA zilizo na uwezo wa kushikamana na mpangilio fulani wa DNA au RNA uliochaguliwa. [6] Katika antisense RNA wao huzuia utengenezwaji wa protini kutoka wa strandi fulani za mjumbe RNA kwa kuzifungama, katika mchakato uitwao hybridization . [12] Oligonucleotidi za Antisense zinaweza kutumika kulenga RNA maalum. Ikiwa ufungamanaji utafanyika, hybrid hii inaweza kuharibiwa na kimeng'enya RNase H . [12] RNase H ni kimeng'enya ambacho hufanya RNA hidrolisisi, na inapotumiwa katika oligonukleotidi ya antisensi husababisha kupungua kwa kutengenezwa kwa protini kutoka kwa mRNA kwa kiwango cha 80-95%. [6]

Janet Heasman ndiye aliyekuwa mwanzilishi wa matumizi ya oligonukleotidi za Morpholino antisensi katika kunyamazisha jeni kwenye wanyama wenye uti wa mgongo, kwa kutumia Xenopus . [13] Oligonukleotidi hizi sasa ni mbinu za kawaida katika developmental biology na zinatumiwa kuchunguza mabadiliko ya usemi na utendakazi wa jeni. Dawa za Morpholino zilizoidhinishwa na FDA ni pamoja na eteplirsen na golodirsen . Oligonukleotidi za antisense pia zimetumiwa kuzuia replication ya virusi vya mafua katika seli. [14] [15]

Magonjwa ya neurodegenerative yanayo sababishwa na protini mutant ni malengo mazuri ya matibabu ya oligonukleotidi antisensi kwa sababu ya uwezo wao wa kulenga na kurekebisha mlolongo maalum wa RNA na kwa uteuzi wa hali ya juu. [3] Magonjwa mengi ya jenetika ikiwa ni pamoja na ugonjwa wa Huntington, ugonjwa wa Alzeheimer's, ugonjwa wa Parkinson, na ugonjwa wa amyotrophic lateral sclerosis (ALS) yamehusishwa na mabadiliko ya DNA ambayo husababisha mfuatano usio sahihi wa RNA na protini zilizotafsiriwa vibaya ambazo huwa na athari mbaya za kifiziolojia. [16]

Uingiaji katika Seli

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Kikwazo kikuu katika ufanisi wa tiba ya oligonukleotidi ni uchukuaji kwenye seli/uingiaji wa oligonukleotidi katika seli . Uingiaji wa moja kwa moja, kama vile wa dawa nyingi za molekuli ndogo, huzuiwa na uti wa mgongo wa polyanionic na ukubwa wa molekuli ya ON. Mbinu kamili za uchukuaji oligonukleotidi na usafirishaji wake ndani ya seli kuelekea mahali lengwa ili kuleta athari ya tiba bado hazijaelezwa kwa kina. Aidha, tofauti chache katika muundo/uboreshaji wa ON (vide supra) na aina tofauti ya seli huwa na athari kubwa katika uingiaji wa seli. Inaaminika kuwa uchukuaji wa seli hufanyika kwa njia tofauti tofauti baada ya adsorption ya ON kwenye surface ya seli. Tafiti zinaonyesha kwamba seli nyingi za tissue culture huchukua ASOs (uhusiano wa phosphorothiote) kwa njia zisizo na manufaa, ambayo matekeo yake ni kukosekana kwa athari ya antisense inayotarajiwa. Hii ni kinyume na muunganisho wa ASO na ligandi zinazotambuliwa na G-coupled receptors ambazo husababisha kuongezeka kwa uchukuzi uliyo na athari tarajiwa . [17] Kando ya uainishaji huo, uingiaji ndani ya seli mara nyingi huendelea kwa njia inayotegemea nishati (receptor mediated endocytosis) lakini passive diffusion isiyo hitaji nishati (=gymnosis) pia huweza kutumika. Baada ya kupita utando wa seli, ON huingia kwenye early endosomes ambazo husafirishwa kuelekea late endosomes ambazo hatimaye huunganishwa na lysosomes zilizo na vimeng'enya vya uharibifu katika pH ya chini. [18] Ili kutekeleza utendakazi wake wa matibabu, ON inahitaji kuepuka endosome kabla ya kuharibiwa . Hadi wa leo hakuna njia ya jumla ya kushinda matatizo ya ufikishaji wa ON katika sehemu lengwa, uchukuaji wa seli na uepukaji kwa endosomes lakini kuna mbinu kadhaa ambazo daima hulengwa kwa seli maalumu (pamoja na receptors zao). [19]

Muunganisho wa tiba za ON kwa huluki inayohusika na utambuzi/uchukuaji wa seli sio tu huongeza uingiaji (vide supra) bali pia huaaminika kupunguza utata wa uchukuaji wa seli kwani utaratibu mmoja (unaojulikana zaidi) huhusika. [18] Hili limeafikiwa na viunganishi vidogo vya molekuli-ON kwa mfano vyenye N-acetyl galactosamine ambayo hulenga receptors za hepatocytes . [20] Viunganishi hivi ni mfano bora wa kupata ongezeko la uchukuaji wa seli uliunganishwa na uwasilishaji kwani receptors huwa nyingi kupita kiasi kwenye seli lengwa (linganisha antibody-drug conjugates ambavyo hutumia receptors zilizonyingi kupita kiasi kwenye seli za saratani). [19] Antibodies zimetumika sana na kufanyiwa utafiti mkubwa kuhusiana na ufikishaji wa ON katika sehemu lengwa na kuongezeka kwa uchukuzi wazo kwenye seli.

Mbinu za uchambuzi

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Chromatografia

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Alkylamides zinaweza kutumika kama chromatographic stationary phases. [21] Awamu hizo zimechunguzwa kwa utenganishaji wa oligonukleotidi. [22] Ion-pair reverse-phase high-performance liquid chromatography hutumiwa kutenganisha na kuchanganua oligonukleotidi baada ya usanisi otomatiki. [23]

Mass spectrometry

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Mchanganyiko wa asidi 5-methoxysalicylic na spermine unaweza kutumika kama matrix kwenye uchanganuzi wa oligonukleotidi katika MALDI mass spectrometry . [24] ElectroSpray Ionization Mass Spectrometry (ESI-MS) pia ni kifaa chenye nguvu katika kubainisha ratili ya oligonucleotidi. [25]

Microarray ya DNA

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DNA microarrays zina matumizi muhimu ya uchambuzi wa oligonukleotidi. Zikilinganishwa na cDNA microarrays, microarrays za oligonukleotidi zina umaalum uliodhibitiwa zaidi kuliko hybridization, na uwezo wa kupima uwepo na kuenea kwa alternatively spliced au poliadenylated sequences. [26] Aina moja ndogo ya DNA microarrays inaweza kuelezewa kuwa substrates (nylon, kioo, nk) ambapo oligonukleotidi zimeshikiliwa kwa msongamano mkubwa. [27] Kuna idadi ya matumizi ya DNA microarrays katika sayansi ya maisha.

Marejeo

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  1. ↑ Yang J, Stolee JA, Jiang H, Xiao L, Kiesman WF, Antia FD, Fillon YA, Ng A, Shi X (Oktoba 2018). "Solid-Phase Synthesis of Phosphorothioate Oligonucleotides Using Sulfurization Byproducts for in Situ Capping". The Journal of Organic Chemistry. 83 (19): 11577–11585. doi:10.1021/acs.joc.8b01553. PMID 30179468. {{cite journal}}: Unknown parameter |displayauthors= ignored (|display-authors= suggested) (help)CS1 maint: date auto-translated (link)
  2. ↑ Qureshi A, Thakur N, Monga I, Thakur A, Kumar M (1 Januari 2014). "VIRmiRNA: a comprehensive resource for experimentally validated viral miRNAs and their targets". Database. 2014: bau103. doi:10.1093/database/bau103. PMC 4224276. PMID 25380780.{{cite journal}}: CS1 maint: date auto-translated (link)
  3. ↑ 3.0 3.1 Monga I, Qureshi A, Thakur N, Gupta AK, Kumar M (2017). "ASPsiRNA: A Resource of ASP-siRNAs Having Therapeutic Potential for Human Genetic Disorders and Algorithm for Prediction of Their Inhibitory Efficacy". G3: Genes, Genomes, Genetics. 7 (9): 2931–2943. doi:10.1534/g3.117.044024. PMC 5592921. PMID 28696921.
  4. ↑ Weiss, B., ed. (1997). Antisense Oligodeoxynucleotides and Antisense RNA : Novel Pharmacological and Therapeutic Agents. Boca Raton, Florida: CRC Press
  5. ↑ Weiss B, Davidkova G, Zhou LW (1999). "Antisense RNA gene therapy for studying and modulating biological processes". Cellular and Molecular Life Sciences. 55 (3): 334–58. doi:10.1007/s000180050296. PMID 10228554.
  6. ↑ 6.0 6.1 6.2 Dias N, Stein CA (Machi 2002). "Antisense oligonucleotides: basic concepts and mechanisms". Molecular Cancer Therapeutics. 1 (5): 347–55. PMID 12489851.{{cite journal}}: CS1 maint: date auto-translated (link)
  7. ↑ Frazier KS (Januari 2015). "Antisense oligonucleotide therapies: the promise and the challenges from a toxicologic pathologist's perspective". Toxicologic Pathology. 43 (1): 78–89. doi:10.1177/0192623314551840. PMID 25385330.{{cite journal}}: CS1 maint: date auto-translated (link)
  8. ↑ 8.0 8.1 8.2 8.3 DeVos SL, Miller TM (Julai 2013). "Antisense oligonucleotides: treating neurodegeneration at the level of RNA". Neurotherapeutics. 10 (3): 486–97. doi:10.1007/s13311-013-0194-5. PMC 3701770. PMID 23686823.{{cite journal}}: CS1 maint: date auto-translated (link)
  9. ↑ 9.0 9.1 Eckstein F (Aprili 2000). "Phosphorothioate oligodeoxynucleotides: what is their origin and what is unique about them?". Antisense & Nucleic Acid Drug Development. 10 (2): 117–21. doi:10.1089/oli.1.2000.10.117. PMID 10805163.{{cite journal}}: CS1 maint: date auto-translated (link)
  10. ↑ Stein CA, Subasinghe C, Shinozuka K, Cohen JS (Aprili 1988). "Physicochemical properties of phosphorothioate oligodeoxynucleotides". Nucleic Acids Research. 16 (8): 3209–21. doi:10.1093/nar/16.8.3209. PMC 336489. PMID 2836790.{{cite journal}}: CS1 maint: date auto-translated (link)
  11. ↑ 11.0 11.1 Michel BY, Dziuba D, Benhida R, Demchenko AP, Burger A (2020). "Probing of Nucleic Acid Structures, Dynamics, and Interactions With Environment-Sensitive Fluorescent Labels". Frontiers in Chemistry (kwa English). 8: 112. Bibcode:2020FrCh....8..112M. doi:10.3389/fchem.2020.00112. PMC 7059644. PMID 32181238.{{cite journal}}: CS1 maint: unrecognized language (link)
  12. ↑ 12.0 12.1 Crooke ST (Aprili 2017). "Molecular Mechanisms of Antisense Oligonucleotides". Nucleic Acid Therapeutics. 27 (2): 70–77. doi:10.1089/nat.2016.0656. PMC 5372764. PMID 28080221.{{cite journal}}: CS1 maint: date auto-translated (link)
  13. ↑ Heasman J, Kofron M, Wylie C (Juni 2000). "Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach". Developmental Biology. 222 (1): 124–34. doi:10.1006/dbio.2000.9720. PMID 10885751.{{cite journal}}: CS1 maint: date auto-translated (link)
  14. ↑ Kumar P, Kumar B, Rajput R, Saxena L, Banerjea AC, Khanna M (Novemba 2013). "Cross-protective effect of antisense oligonucleotide developed against the common 3' NCR of influenza A virus genome". Molecular Biotechnology. 55 (3): 203–11. doi:10.1007/s12033-013-9670-8. PMID 23729285.{{cite journal}}: CS1 maint: date auto-translated (link)
  15. ↑ Kumar B, Khanna M, Kumar P, Sood V, Vyas R, Banerjea AC (Mei 2012). "Nucleic acid-mediated cleavage of M1 gene of influenza A virus is significantly augmented by antisense molecules targeted to hybridize close to the cleavage site". Molecular Biotechnology. 51 (1): 27–36. doi:10.1007/s12033-011-9437-z. PMID 21744034.{{cite journal}}: CS1 maint: date auto-translated (link)
  16. ↑ Smith RA, Miller TM, Yamanaka K, Monia BP, Condon TP, Hung G, Lobsiger CS, Ward CM, McAlonis-Downes M, Wei H, Wancewicz EV, Bennett CF, Cleveland DW (Agosti 2006). "Antisense oligonucleotide therapy for neurodegenerative disease". The Journal of Clinical Investigation. 116 (8): 2290–6. doi:10.1172/JCI25424. PMC 1518790. PMID 16878173. {{cite journal}}: Unknown parameter |displayauthors= ignored (|display-authors= suggested) (help)CS1 maint: date auto-translated (link)
  17. ↑ Ming, Xin; Alam, Md Rowshon; Fisher, Michael; Yan, Yongjun; Chen, Xiaoyuan; Juliano, Rudolph L. (2010-06-15). "Intracellular delivery of an antisense oligonucleotide via endocytosis of a G protein-coupled receptor". Nucleic Acids Research. 38 (19): 6567–6576. doi:10.1093/nar/gkq534. ISSN 1362-4962.
  18. ↑ 18.0 18.1 Hawner, Manuel; Ducho, Christian (2020-12-16). "Cellular Targeting of Oligonucleotides by Conjugation with Small Molecules". Molecules. 25 (24): 5963. doi:10.3390/molecules25245963. ISSN 1420-3049.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  19. ↑ 19.0 19.1 Crooke, S. T. (2017). "Cellular uptake and trafficking of antisense oligonucleotides". Nat Biotechnol. 35 (3): 230–237.
  20. ↑ Prakash, Thazha P.; Graham, Mark J.; Yu, Jinghua; Carty, Rick; Low, Audrey; Chappell, Alfred; Schmidt, Karsten; Zhao, Chenguang; Aghajan, Mariam (Julai 2014). "Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice". Nucleic Acids Research. 42 (13): 8796–8807. doi:10.1093/nar/gku531. ISSN 1362-4962. PMC 4117763. PMID 24992960.{{cite journal}}: CS1 maint: date auto-translated (link)
  21. ↑ Buszewski B, Kasturi P, Gilpin RK, Gangoda ME, Jaroniec M (Agosti 1994). "Chromatographic and related studies of alkylamide phases". Chromatographia. 39 (3–4): 155–61. doi:10.1007/BF02274494.{{cite journal}}: CS1 maint: date auto-translated (link)
  22. ↑ Buszewski B, Safaei Z, Studzińska S (Januari 2015). "Analysis of oligonucleotides by liquid chromatography with alkylamide stationary phase". Open Chemistry. 13 (1). doi:10.1515/chem-2015-0141.{{cite journal}}: CS1 maint: date auto-translated (link)
  23. ↑ Gilar, M.; Fountain, K. J.; Budman, Y.; Neue, U. D.; Yardley, K. R.; Rainville, P. D.; Russell Rj, 2nd; Gebler, J. C. (2002-06-07). "Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides:: Retention prediction". Journal of Chromatography A (kwa Kiingereza). 958 (1–2): 167–182. doi:10.1016/S0021-9673(02)00306-0. ISSN 0021-9673. PMID 12134814.{{cite journal}}: CS1 maint: numeric names: authors list (link)
  24. ↑ Distler AM, Allison J (Aprili 2001). "5-Methoxysalicylic acid and spermine: a new matrix for the matrix-assisted laser desorption/ionization mass spectrometry analysis of oligonucleotides". Journal of the American Society for Mass Spectrometry. 12 (4): 456–62. Bibcode:2001JASMS..12..456D. doi:10.1016/S1044-0305(01)00212-4. PMID 11322192.{{cite journal}}: CS1 maint: date auto-translated (link)
  25. ↑ Shah S, Friedman SH (Machi 2008). "An ESI-MS method for characterization of native and modified oligonucleotides used for RNA interference and other biological applications". Nature Protocols. 3 (3): 351–6. doi:10.1038/nprot.2007.535. PMID 18323805.{{cite journal}}: CS1 maint: date auto-translated (link)
  26. ↑ Relógio A, Schwager C, Richter A, Ansorge W, Valcárcel J (Juni 2002). "Optimization of oligonucleotide-based DNA microarrays". Nucleic Acids Research. 30 (11): 51e–51. doi:10.1093/nar/30.11.e51. PMC 117213. PMID 12034852.{{cite journal}}: CS1 maint: date auto-translated (link)
  27. ↑ Gong P, Harbers GM, Grainger DW (Aprili 2006). "Multi-technique comparison of immobilized and hybridized oligonucleotide surface density on commercial amine-reactive microarray slides". Analytical Chemistry. 78 (7): 2342–51. doi:10.1021/ac051812m. PMID 16579618.{{cite journal}}: CS1 maint: date auto-translated (link)

Kusoma zaidi

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  • Spingler B (Januari 2012). "Chapter 3. Metal-Ion-Promoted Conformational Changes of Oligonucleotides". Katika Sigel A, Sigel H, Sigel RK (whr.). Interplay between metal ions and nucleic acids. Juz. la 10. Springer Science & Business Media. ku. 103–118. doi:10.1007/978-94-007-2172-2_3. PMID 22210336. {{cite book}}: |journal= ignored (help)CS1 maint: date auto-translated (link)

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